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Friday, 7 September 2018

Chromatography is defined as a
separation technique of a mixture, which is based on the interactions of the
components with two phases i.e. mobile and stationary phase, as the mixture is
allowed to run through a supporting medium.

Definitions:

Mobile phase: Mobile phase is the solvent which is made to
run through the supporting medium.

Stationary Phase: This is a coating or layer on the
supporting medium which remains stationary and allowed to interact with the
components of the mixture.

Supporting medium: It is the solid surface on which stationary
phase is bonded.

Classification of Chromatography

Most common basis of the classification of the chromatography
is based on the type of mobile phase.

Type of chromatography

Type of mobile phase

Gas Chromatography (GC)

Gas

Liquid Chromatography (LC)

Liquid

Further these chromatography techniques can be classified on
the basis of the stationary phases used in these techniques:

GAS CHROMATOGRAPHY

Gas Chromatography technique

Type of Stationary Phase

Gas Solid Chromatography

Solid

Gas Liquid Chromatography

Liquid

Bonded Phase Chromatography

Chemically
derivatized solid support

LIQUID CHROMATOGRAPHY

Liquid Chromatography technique

Type of Stationary Phase

Adsorption Chromatography

Solid
underivatized support

Ion Exchange Chromatography

Fixed
charges on a solid support

Partition Chromatography

Liquid
coated support

Size Exclusion Chromatography

Porous
support

Affinity Chromatography

Immobilized
ligands with support

The overall classification of the chromatography techniques
can be summed up as follows:

Primary Classification

Secondary Classification

Stationary Phase

Types of Equilibrium

Liquid Chromatography

Liquid-Liquid

Liquid
supported on solid

Partition

Liquid-Solid

Solid

Adsorption

Liquid-bonded
phase

Organic
groups bonded to solid surface

Partition
between liquid & Bonded phase

Ion-Exchange

Resins
with charged species

Ion
Exchange

Size
Exclusion

Polymers
having liquids in the voids

Partitioning

Gas
Chromatography

Gas-Liquid

Liquid
on solid

Partition

Gas-Solid

Solid

Adsorption

Gas-Bonded
Phase

Organic
species bonded on solid surface

Partition
between liquid & Bonded phase

Superficial Fluid Chromatography

Organic
species bonded on solid surface

Partition
between Super-critical fluid & Bonded phase

THOERY
OF CHROMATOGRAPHY

The theory of the chromatography can be best
understood with the following points:

1 1. Chromatogram:
Chromatogram is the graph between the volume of the analyte eluted versus the
time taken for the elution.

Basic Chromatogram

a

2. Separation
of the solute: Separation of the solute in the chromatographic system depends
on the two factors:

a.Solute
retention: Solute retention or retention time/retention volume best describes
the interaction of the solute with the stationary and the mobile phase.

·In a particular system, the retention of
solute depends upon the length of the column & the flow rate of the mobile
phase.

Average migration rate,
µ = L (Column Length)/ tR

·Capacity factor (k’): Capacity factor is
more acceptable variable for studying the solute retention as it is independent
of the column length & flow rate.

K’ = (tR – tM)/tM
or (VR-VM)/VM

·In the simplest definition, K’ can be
defined as:

K’: Moles of A in stationary phase/
Moles of A in Mobile phase

K’ ≤ 1, separation is poor

K’ > 30, separation is poor

K’ = 2-10, separation is optimum

b. Efficiency
of the chromatography technique: Efficiency is the term used to define how
efficiently an chromatography technique can separate the mixture of compounds.

·It is dependent on the width of the
peak.

·Narrow the peak more efficient will be
the technique.

·Mathematically, efficiency is measured
in the terms of the Number of theoretically plates (N),

3.Broadening of the peaks: Broadening of the
peak in a chromatogram is attributed to the following factors:

a.Eddy’s
diffusion: This occurs due to the different path traveled by the solute
molecules column.

b.Mobile
phase mass transfer: Mobile phase transfer causes for the broadening of peaks
due to the different travel path of the mobile phase when travels through the
support material of the column.

The
degree of broadening due to the eddy’s diffusion & Mobile phase transfer
depends upon:

·Size of the particles of material.

·Diffusion rate of the solute.

c.Stagnant
mobile phase mass transfer: Sometimes a mobile phase get stagnated in the
support column resulting in the difference in the retention time of the solute
and ultimately the broadening of the peaks.

d.Stationary
phase mass transfer: This is attributed due to the different time spend by the
solute molecule is stationary phase and the stagnant mobile phase resulting in
the broadening of the peak.

e.Longitudinal
diffusion: Broadening of the peak is also attributed due to the diffusion of
the solute molecules while travelling through the column.

Van-Deemter
equation

It relates the linear velocity/flow rate to the
Height equivalent of the theoretical plates (H).